The overall aim of this application is to better understand the role of the fasting milieu in influencing the ability of the ?-cell to respond to subsequent meal challenges. Prediabetes is the transitory state between normal glucose metabolism and type 2 diabetes. Prediabetes is often categorized using fasting glucose concentrations as well as glucose concentrations 2 hours after ingestion of 75g of glucose. These sub-groups differ in their fasting glucose and free fatty acid concentrations, their ?-cell function and their risk of progression to type 2 diabetes. Intriguingly, the component of ?-cell response to glucose that depends on fasting insulin synthesis and storage seems to be impaired in subjects with high free fatty acid concentrations and hyperglycemia. This suggests that elevation of these substrates might directly influence ?-cell function by altering fasting insulin synthesis and, perhaps, drive the progression of prediabetes to diabetes. In addition, there is a unique sub- group of prediabetes with isolated, impaired fasting glucose. Data available to date suggest that these subjects behave like patients with a mutation in the glucokinase (GCK) gene. Previously, our group demonstrated that people with diabetes have hepatic glucokinase dysfunction. In this series of experiments we will ascertain if this subgroup does indeed exhibit impaired glucose sensing alone or in combination with a global defect in ?- cell function. This will help to determine if defects in fasting and postprandial insulin secretion, in response to hyperglycemia, develop independently. Since substrate excess increases demand on the synthetic machinery of the ?-cell, it increases the rate of protein misfolding and induces a protective mechanism, known as the unfolded protein response, intended to restore endoplasmic reticulum homeostasis. The in vivo correlate of this response is unknown ? 1st phase insulin is absent in type 2 diabetes and there is an increase in proinsulin release implying defects in insulin synthesis. However, concentrations of proinsulin are unlikely to be informative of prandial ?-cell function as its clearance kinetics are unknown. The proposed experiments will elucidate how changes in fasting FFA and glucose alter insulin and proinsulin secretion. Since elevated proinsulin has been associated with ?-cell dysfunction, we will examine how changes in proinsulin secretion change over time. The proposed experiments will help elucidate the mechanisms by which fasting substrate excess contributes to ?-cell dysfunction, and to the progression of prediabetes to type 2 diabetes.